Figure 4

CQ potentiates the accumulation of LC3-II induced by 5-FU. (A) The expression of LC3-II was quantified by Western blot in HT-29 cells treated with 5-FU alone (left) for 48 h or after 12-h pretreatment with CQ (right). Treatment of HT-29 cells with 5-FU alone resulted in increased accumulation of LC3-II time-dependently, and the effect was potentiated by CQ-pretreatment. The expression levels of LC3-II are expressed as the density measured by the Image J software, standardized by the density of β-actin. (B) The intracellular localization of LC3 was analyzed by immunofluorescence microscopy, by staining the cells with fluorescent antibodies to LC3. Control (untreated) and CQ-treatment cells exhibited a weak and diffuse cytoplasmic staining with LC3-associated green fluorescence, whereas those treated with 5-FU and CQ pre-treatment + 5-FU exhibited an evident punctuate green fluorescence pattern of LC3, which is a typical feature of LC3 distribution within autophagosomes (LC3-II).